African trypanosomes cause disease and suffering in vast regions of sub-Saharan Africa and the drugs available to eliminate the disease are sub-optimal, to say the least. Many laboratories, including our own, have been engaged in studying the biology of Trypanosoma brucei and have contributed to the understanding of several mechanisms that are unique to trypanosomes. The discovery of trypanosome-specific pathways is the first step towards identifying new potential drug targets, which may be exploited against the parasite itself. Since 1998 when we discovered the RNAi pathway in Trypanosoma brucei our research has focused towards understanding the mechanism and components of RNAi. These studies are essential to eventually attempt to use small RNAs as drugs against trypanosomes. This possibility is not without foundation, since there are already several clinical trials in place to use small RNAs to cure certain human diseases. The experiments we propose stem directly from three main findings generated during the last funding period. First, sequencing of small interfering RNAs (siRNAs), from both bloodstream and procyclic trypanosomes has revealed a new target of the RNAi pathway, namely a family of satellite-like repeats (CIR147) located at chromosomal internal sites. These repeats may function in some aspect of chromosome structure and/or regulation of gene expression. Second, we have identified the RNAi genes in Leishmania braziliensis and in collaboration with Steve Beverley have demonstrated that the RNAi pathway is operational. However, L brasiliensis has some limitations for studying pathogenesis because at present there is no suitable small animal model of the disease. In contrast, Diane McMahon-Pratt at Yale has established a mouse model for L. panamensis, a close relative of L. brasiliensis, which also has the RNAi genes. Thus, our findings open the way to establish tools for applying the wonders of RNAi technology to the study of different aspects of the biology and pathogenesis of Leishmania. Third, we have discovered that a second T. brucei Argonaute-like protein (termed PiwiS) is localized in the mitochondrion and forms foci reminiscent of the antipodal sites where some enzymes for kinetoplast DNA (kDNA) replication localize, implicating PiwiS in some aspect of kDNA biogenesis. In the last few years Argonaute proteins have emerged as central regulators of an outstandingly wide range of phenomena, both at the transcriptional and post-transcriptional levels. Thus, we hypothesize that PiwiS functions in some aspect of RNA silencing in the mitochondrion. It should be noted that this protein is the first example of an Argonaute protein that localizes to an organelle. Our specific aims for the next funding period are: 1. To further mine the biology of trypanosomatids through high-throughput sequencing of small RNAs from T. brucei and from L. braziliensis, and through the functional and structural characterization of the CIR147 satellite-like repeat family. 2. To establish tools for heritable and regulated RNAi in L. panamensis. 3. To establish the function of PiwiS in the T. brucei mitochondrion. [unreadable] [unreadable] Project Narrative: African trypanosomes cause disease and suffering in vast regions of sub-Saharan Africa and the drugs available to eliminate the disease are sub-optimal, to say the least. Our research has focused towards understanding the mechanism and components of RNA interference to eventually attempt to use small RNAs as drugs against trypanosomes. This possibility is not without foundation, since there are already several clinical trials in place to use small RNAs to cure certain human diseases. [unreadable] [unreadable] [unreadable]